Deletion of the K(V)1.1 potassium channel causes epilepsy in mice.

Mice lacking the voltage-gated potassium channel alpha subunit, K(V)1.1, display frequent spontaneous seizures throughout adult life. In hippocampal slices from homozygous K(V)1.1 null animals, intrinsic passive properties of CA3 pyramidal cells are normal. However, antidromic action potentials are recruited at lower thresholds in K(V)1.1 null slices. Furthermore, in a subset of slices, mossy fiber stimulation triggers synaptically mediated long-latency epileptiform burst discharges. These data indicate that loss of K(V)1.1 from its normal localization in axons and terminals of the CA3 region results in increased excitability in the CA3 recurrent axon collateral system, perhaps contributing to the limbic and tonic-clonic components of the observed epileptic phenotype. Axonal action potential conduction was altered as well in the sciatic nerve--a deficit potentially related to the pathophysiology of episodic ataxia/myokymia, a disease associated with missense mutations of the human K(V)1.1 gene.

Paroxysmal afterpotentials and role of calcium-dependent potassium conductivity in neuronal activity of strychninized neocortex.

Reactions of cortical suprasylvian gyrus neurons were investigated intracellularly after supracortical strychnine application in immobilized and anaesthetized cats. It was shown that paroxysmal depolarizing shifts of membrane potential could be accompanied by de- and hyperpolarizing afterpotentials. When passing from epileptiform to normal physiological activity, short afterhyperpolarizations, 300-500 ms in duration, were converted into inhibitory postsynaptic potentials which were also accompanied by a decrease in membrane potential. When the frequency of paroxysmal discharge was less than 1 s, prolonged (1-2 s) afterhyperpolarizations were observed; at a higher frequency their summation led to tonic hyperpolarization of the membrane. The ictal discharges were accompanied by postictal hyperpolarizations of up to 30 s duration. The intracellular injection of EGTA blocking Ca2(+)-dependent potassium conductivity eliminated prolonged after- and postictal hyperpolarizations and produced depolarizing afterpotentials and a gradual depolarization of cell membranes. Our results indicate that the development of short hyperpolarizing afterpotentials could be determined by the inhibitory synaptic effects. The activation of Ca2(+)-dependent potassium conductivity caused by the development of prolonged afterhyperpolarizations and postictal polarizations, as well as maintained tonic hyperpolarization of cell membranes. Obviously, the depolarizing afterpotentials are of a non-synaptic origin and can be induced by inward calcium current.

Lack of vesicular zinc in mossy fibers does not affect synaptic excitability of CA3 pyramidal cells in zinc transporter 3 knockout mice.

Zinc is found throughout the CNS in synaptic vesicles of glutamatergic neurons and has been suggested to have a modulatory role in the brain because of its interaction with voltage- and ligand-gated ion channels. We took advantage of zinc transporter 3 knockout mice, which lack vesicular zinc, to study the possible physiological role of this heavy metal in hippocampal mossy fiber neurotransmission. We examined postsynaptic responses evoked by mossy fiber activation, recorded in CA3 pyramidal cells in hippocampal slices prepared from zinc transporter 3 knockout and wild-type mice. Field-potential response threshold and amplitude, input-output curves, and paired-pulse evoked responses were the same in slices from zinc transporter 3 knockout and wild-type mice. Furthermore, neither amplitude nor duration of pharmacologically isolated N-methyl-D-aspartate, non-N-methyl-D-aspartate, GABA(A), and GABA(B) receptor-mediated postsynaptic potentials differed between zinc transporter 3 knockout and wild-type mice. There was no difference in the magnitude of epileptiform discharges evoked by repetitive stimulation or kainic acid application. However, in slices from zinc transporter 3 knockout mice, there was greater attenuation of GABA(A)-mediated inhibitory postsynaptic potentials during tetanic stimulation compared with slices from wild-type animals. We conclude that lack of vesicular zinc in mossy fibers does not significantly affect the mossy fiber-associated synaptic excitability of CA3 pyramidal cells; however, zinc may modulate GABAergic synaptic transmission under conditions of intensive activation.

Reverberation of chloride-dependent synaptic potentials in the rat entorhinal cortex in vitro.

The spontaneous activity generated by rat entorhinal neurons during application of 4-aminopyridine (4AP; 50 microM) was studied with intracellular and extracellular field-potential recordings in an vitro slice preparation. Long-lasting depolarizations (LLDs) with amplitudes of 15 +/- 7.6 mV (mean +/- SD; n = 14) and durations of 1.65 +/- 0.77 s (n = 14) occurred at 0.036 +/- 0.01/s (n = 14). Each LLD was followed by a rhythmic sequence of depolarizing potentials (up to 22 events) with amplitudes of 4-30 mV, durations of 40-500 ms and frequency of 0.9 +/- 0.2/s (n = 14). These intracellular potentials were mirrored by negative-going field potentials, suggesting that they represented synchronous events. Membrane input resistance decreased by 79-86% during both LLDs and subsequent rhythmic depolarizations. Intracellular injection of steady depolarizing or hyperpolarizing current modified the amplitude of these potentials in a similar manner: the reversal potential of the LLDs and of the rhythmic depolarizations was -66.4 +/- 4 mV and -67.9 +/- 3.2 mV, respectively (n = 7). Intracellular injection of Cl- increased the amplitude of both types of potentials. Spontaneous LLDs continued to occur during application of the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (10 microM), a procedure that abolished the subsequent rhythmic depolarizations (n = 3). LLDs were blocked by further addition of the gamma-aminobutyric acid (GABA)A receptor antagonist bicuculline methiodide (10 microM, n = 3). Our findings demonstrate that during 4AP application entorhinal neurons generate glutamatergic-independent LLDs as well as synchronous, Cl(-)-dependent depolarizations that reverberate through non-NMDA-mediated excitatory circuits.

[Strychnine-induced changes of the membrane and postsynaptic potentials in neocortical neurons]. / Izmeneniia membrannogo i postsinapticheskikh potentsialov neironov novoi kory, vyzvannye strikhninom.

Intracellular responses of neurons of the suprasylvian gyrus to the intracortical stimulation (ICS) before and after superficial application of strychnine were investigated in experiments on immobilized and unanaesthetized cats. The normal cortex neurons reacted to ICS by monosynaptic EPSPs followed by IPSPs. Strychnine application triggered the epileptiform activity and appearance in neurons of paroxysmal depolarization shifts of the membrane potential (MP) which were replaced by hyperpolarization potentials. An increase and summation of the latter elicited the MP enlargement and either reduction or suppression of background spike activity. Intracellular injections of EGTA blocking the membrane calcium-dependent potassium conductivity (gK(Ca)) have eliminated the hyperpolarization potentials. Development of epileptiform activity was accompanied by depression of IPSPs and increase of the monosynaptic EPSPs. The contribution of gK(Ca) and of postsynaptic inhibition to the epileptogenesis is discussed.

Participation of GABAA-mediated inhibition in ictallike discharges in the rat entorhinal cortex.

The spontaneous, synchronous activity induced by 4-aminopyridine (4AP, 50 microM) in the adult rat entorhinal cortex was analyzed with simultaneous field potential and intracellular recordings in an in vitro slice preparation. Four-AP induced isolated negative-going field potentials (interval of occurrence = 27.6 +/- 9.9 (SD) s; n = 27 slices) that corresponded to intracellular long-lasting depolarizations (LLDs), and ictallike epileptiform discharges (interval of occurrence = 10.4 +/- 5.7 min; n = 27 slices) that were initiated by the negative field potentials. LLDs recorded with K-acetate-filled microelectrodes triggered few action potentials of variable amplitude and had a duration of 1.7 +/- 0.8 s (n = 26 neurons), a peak amplitude of 11.8 +/- 5.0 mV (n = 26 neurons) and a reversal potential of -66.2 +/- 3.9 mV (n = 17 neurons). The ictal discharges studied with K-acetate microelectrodes consisted of prolonged depolarizations (duration = 72.9 +/- 44.3 s; peak amplitude = 29.2 +/- 11.4 mV; n = 25 neurons) with action-potential firing during both the tonic and the clonic phase. These depolarizations had a reversal potential of -45.3 +/- 3.8 mV (n = 4 neurons). Intracellular Cl- diffusion from KCl-filled microelectrodes made both LLDs and ictal depolarizations increase in amplitude (30.5 +/- 8.2 mV, n = 8 and 41.8 +/- 9.8 mV, n = 6 neurons, respectively). LLDs recorded with KCl and 2-(trimethyl-amino)N-(2, 6-dimethylphenyl)-acetamide (QX-314) microelectrodesreached an amplitude of 36.3 +/- 5.2 mV, lasted 12.5 +/- 6.5 s, and had a reversal potential of -31.3 +/- 2.5 mV (n = 4 neurons); under these recording procedures the ictal discharge amplitude was 41.5 +/- 5.0 mV and the reversal potential -24.0 +/- 7.0 mV (n = 4 neurons). The N-methyl-D-aspartate (NMDA) receptor antagonist 3,3-(2-carboxy-piperazine-4-yl)-pro-pyl-l-phosphonate (10 microM, n = 5 neurons) alone or concomitant with the nonNMDA receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (10 microM, n = 4 neurons) abolished ictal discharges, without influencing LLDs. LLDs were blocked by the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline methiodide (BMI, 10 microM, n = 6 neurons) or the mu-opioid receptor agonist (-Ala2-N-Me-Phe, Gly-ol) enkephalin (DAGO, 10 microM, n = 2 neurons). Application of BMI (n = 4 neurons) or DAGO (n = 2 neurons) to control the medium abolished LLDs and ictal discharges but disclosed a novel type of epileptiform depolarization that lasted 3.5 +/- 1.2 s and occurred every 5.2 +/- 2.6 s (n = 6 neurons). Our data indicate that 4AP induces in the rat entorhinal cortex a synchronous, GABA-mediated potential that is instrumental in initiating NMDA-dependent, ictal discharges. Moreover we present evidence for an active role played by GABAA-mediated potentials in the maintenance and termination of these prolonged epileptiform events.

Laminar organization of epileptiform discharges in the rat entorhinal cortex in vitro.

1. Interictal and ictal epileptiform discharges induced by 4-aminopyridine (4AP, 50 microM) were studied in the rat lateral entorhinal cortex with field potential and intracellular recordings in an in vitro slice preparation. Both types of discharge disappeared in layer II, but continued to occur in layers IV-VI after a knife cut separation was made at approximately 600 micro(m) from the pia (n = 4 slices). 2. Interictal depolarizations recorded in layer IV-VI cells (amplitude, 29.4 +/- 8.6 mV; duration, 386 +/- 177.4 ms, means +/- s.d.; n = 17) were capped by action potential bursts, while smaller interictal depolarizations in layer II cells (amplitude, 11.7 +/- 5.8 mV; duration, 192.6 +/- 47.9 ms; n = 10) were associated with single action potentials and were terminated by a hyperpolarization. Ictal discharges were initiated by an interictal discharge; they were characterized by a depolarization of 31.5 +/- 6.2 mV (n = 12) in layer IV-VI and 11.6 +/- 3.5 mV (n = 7) in layer II neurones. 3. Slow, presumptive Ca2+-mediated spikes occurred in layer II (n = 4) and IV-VI (n = 6) cells loaded with the Na+ channel blocker QX-314 (50 mM). These events were synchronized with population spikes during interictal and ictal discharges, and were abolished by Ni2+ (1 mM, n = 4 cells) along with the 4AP-induced synchronous activity. 4. The N-methyl-D-aspartate (NMDA) receptor antagonist 3, 3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonate (CPP, 10 microM) abolished ictal discharges and reduced interictal depolarizations in layer IV-VI neurones (n = 4). The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) abolished both interictal and ictal activity (n = 4 cells). 5. These findings provide evidence for a role played by NMDA-mediated mechanisms in the generation of epileptiform discharges in the entorhinal cortex. Lack of an NMDA-mediated component along with presence of inhibition in layer II neurones results in attenuation of epileptiform activity at this site. Moreover Ca2+-mediated spikes may contribute to the appearance of epileptiform discharges in this model.

GABA(A)-dependent chloride influx modulates reversal potential of GABA(B)-mediated IPSPs in hippocampal pyramidal cells.

Changes in intracellular chloride concentration, mediated by chloride influx through GABA(A) receptor-gated channels, may modulate GABA(B) receptor-mediated inhibitory postsynaptic potentials (GABA(B) IPSPs) via unknown mechanisms. Recording from CA3 pyramidal cells in hippocampal slices, we investigated the impact of chloride influx during GABA(A) receptor-mediated IPSPs (GABA(A) IPSPs) on the properties of GABA(B) IPSPs. At relatively positive membrane potentials (near -55 mV), mossy fiber--evoked GABA(B) IPSPs were reduced (compared with their magnitude at -60 mV) when preceded by GABA(A) receptor--mediated chloride influx. This effect was not associated with a correlated reduction in membrane permeability during the GABA(B) IPSP. The mossy fiber--evoked GABA(B) IPSP showed a positive shift in reversal potential (from -99 to -93 mV) when it was preceded by a GABA(A) IPSP evoked at cell membrane potential of -55 mV as compared with -60 mV. Similarly, when intracellular chloride concentration was raised via chloride diffusion from an intracellular microelectrode, there was a reduction of the pharmacologically isolated monosynaptic GABA(B) IPSP and a concurrent shift of GABA(B) IPSP reversal potential from -98 to -90 mV. We conclude that in hippocampal pyramidal cells, in which "resting" membrane potential is near action potential threshold, chloride influx via GABA(A) IPSPs shifts the reversal potential of subsequent GABA(B) receptor--mediated postsynaptic responses in a positive direction and reduces their magnitude.

[Responses of neurons of an isolated cortical strip in a state of convulsive excitation to single electrical stimuli]. / Reaktsii neironov izolirovannoi poloski kory golovnogo mozga, nakhodiashchikhsia v sostoianii sudorozhnogo vozbuzdeniia, na odinochnye élektricheskie razdrazheniia.

Responses of isolated cortical slab neurons to single stimuli before, during and after the development of epileptiform state in a slab were investigated in experiments on immobilized and locally anaesthetized cats. It was shown that during the development of generalized seizure activity in an isolated cortical slab its neurons generate EPSP and paroxysmal depolarizing shifts (PDS) of the membrane potential (MP) accompanied by refractory periods. Refractory periods coincide with PDS plato and MP repolarizing shifts. During these shifts single electrical stimuli produce gradually transforming PDS. After cessation of the ictal activity neurons are still able to generate PDS to single stimuli for some time. It is suggested that the role of postsynaptic responses in genesis of the epileptiform activity is not the most important. Nonsynaptic factors are, probably, involved in its generation.

[Prolonged depolarizing potentials of the neurons in a strychninized isolated strip of the cat cerebral cortex]. / Dlitel'nye depoliarizatsionnye potentsialy neironov strikhninizirovannoi izolirovannoi poloski kory mozga koshki.

Reactions of isolated cortical slab neurons to the supracortical application of strychnine were investigated with intracellular registration in experiments on unanaesthetized and immobilized cats. It was shown that some neurons demonstrated prolonged depolarizing potentials (PDP) spontaneously and as reactions to single intracortical electrical stimuli. The development of these potentials could be a result of transformation of the reaction of the "paroxysmal depolarizing shift (PDS)--hyperpolarization" type, where hyperpolarizations were replaced by depolarizing potentials. A gradual increase of depolarizing afterpotentials resulted in DDP generation. These transformations, as a rule, were accompanied by amplification of the summary epileptiform activity in an isolated cortical slab. The suggestion was made that the DDP generation was determined by an increase in the Ca(++)-conductance of the neuronal membrane in an isolated cortical slab with the intensification of paroxysmal reactions.

[The cellular reactions of a strychninized isolated strip of the cat cerebral cortex]. / Reaktsii kletok strikhninizirovannoi izolirovannoi poloski kory golovnogo mozga koshki.

Reactions of neuronal and glial cells of an isolated cortical slab to direct electrical stimulation after supracortical strychnine application were investigated in experiments on immobilized and unanaesthetized cats. Strychnine evoked single epileptiform discharges and afterdischarges in the isolated cortical slab and large paroxysmal depolarization shifts (PDS) of the membrane potential (MP) in the neurons. It was shown that spontaneous summary epileptiform discharges and cellular activity of neurons investigated were synchronized slightly. Electrical stimuli produced a generalized paroxysmal activity in the isolated slab. Neuronal PDSs were accompanied by refractory periods which development did not depend on the MP level. Strychnine enhanced a number of neurons with the background activity in which PDS were generated by rhythmic depolarizing MP oscillations of the nonsynaptic origin. It was shown also that epileptiform reactions of the strychninized isolated cortical slab to the single stimuli were accompanied by large depolarization shifts of the glial cells' MP. The suggestion is made that the paroxysmal excitation development in the strychninized isolated cortical slab was determined by non-synaptic factors and was strongly related to the changes of the extracellular potassium concentration.

[Electrical activity of the neurons in an epileptic focus created in an isolated strip of cerebral cortex by electrical stimulation]. / Elektricheskaia aktivnost' neironov sudorozhnogo ochaga, sozdannogo v izolirovannoi poloske kory mozga élektricheskoi stimuliatsiei.

The peculiarities of the activity of cortical isolated slab neurons were investigated in experiments on unanaesthetized and immobilized cats during the development of seizure spikes evoked by repetitive powerful stimulation. It was shown that in the investigated focus of epileptiform discharges the neurons were not differentiated by the degree of pathological alterations, since paroxysmal membrane potential shifts of all neurons were recorded intracellularly. All these neurons were characterized by a lack of the spike activity. At the same time bursting spike discharges of isolated slab neurons were recorded extracellularly and they did not propagate to the soma. Simultaneous extra- and intracellular recordings of the activity from the same neurons have shown that during the epileptiform activity action potentials were generated in some trigger zones without propagating to the cell bodies. Possible mechanisms of the origin of the spike activity in isolated slab neurons during the development of generalized epileptiform state are discussed.

GABAA-Dependent chloride influx modulates GABAB-mediated IPSPs in hippocampal pyramidal cells.

The relationship between postsynaptic inhibitory responses [the fast GABA(A)-mediated inhibitory postsynaptic potential (IPSP) and the slow GABA(B)-mediated IPSP] were investigated in hippocampal CA3 pyramidal cells. Mossy fiber-evoked GABA(B)-mediated IPSPs were, paradoxically, of greater amplitude in cells with resting membrane potential of -62 mV (13.6 +/- 0.5 mV; mean +/- SE) as compared with cells with resting membrane potential of -54 mV (7.0 +/- 0.8 mV). In addition, when a cell's membrane potential was artificially manipulated, GABA(B)-mediated IPSPs were reduced at relatively depolarized levels (-55 mV) and enhanced at relatively hyperpolarized potentials (at least -60 mV). In contrast, the preceding GABA(A)-mediated IPSPs were larger at the more positive membrane potentials and smaller as the cell was hyperpolarized. Similar voltage dependency was obtained when monosynaptic GABA(A)- and GABA(B)-mediated IPSPs were isolated in the presence of glutamatergic receptor antagonists. However, monosynaptic GABA(B)-mediated IPSPs isolated in the presence of glutamatergic and GABA(A) receptor antagonists were not reduced at the more positive membrane potentials, and were significantly larger in amplitude than GABA(B)-mediated IPSPs preceded by a monosynaptic GABA(A)-mediated IPSP. The amplitude of the isolated monosynaptic GABA(B)-mediated IPSPs recorded with potassium chloride-containing microelectrodes was significantly smaller than the comparable potential recorded with potassium acetate microelectrodes without chloride. We conclude that voltage-dependent chloride influx, via GABA(A) receptor-gated channels, modulates postsynaptic GABA(B)-mediated inhibition in hippocampal CA3 pyramidal cells.

[The postsynaptic components of the paroxysmal reactions of the neurons in the strychninized neocortex]. / Postsinapticheskie komponenty paroksizmal'nykh reaktsii neironov strikhninizirovannoi novoi kory mozga.

Intracellular responses of neurons of cortical strychninized suprasylvian gyrus were investigated in experiments on immobilized and unanaesthetized cats. Paroxysmal depolarizing shifts (PDS) of the neuronal membrane potential were registered. They consisted of the burst discharge (BD) and slow depolarization wave. By means of intracellular stimulation it was shown that PDS could be summarized and were able to change their form and size. BD in PDS were triggered by large EPSPs which could be elicited from paroxysmal responses. Presumably, the intradendritic recordings have shown the presence of large EPSPs during generation of epileptiform discharges in the neocortex. In some neurons PDSs were accompanied by hyperpolarizing potentials which were apparently IPSPs because they were reversed at the intracellular Cl- injections. The contribution of excitatory and inhibitory synaptic influences to the neuronal paroxysmal responses is discussed.

Synchronous GABA-mediated potentials and epileptiform discharges in the rat limbic system in vitro.

Application of 4-aminopyridine (4AP, 50 microM) to combined slices of adult rat hippocampus-entorhinal cortex-induced ictal and interictal epileptiform discharges, as well as slow field potentials that were abolished by the mu-opioid agonist [D-Ala2,N-Me-Phe4,Gly-ol5] enkephalin (DAGO, 10 microM) or the GABAA receptor antagonist bicuculline methiodide (BMI, 10 microM); hence, they represented synchronous GABA-mediated potentials. Ictal discharges originated in the entorhinal cortex and propagated to the hippocampus, whereas interictal activity of CA3 origin was usually recorded in the hippocampus. The GABA-mediated potentials had no fixed site of origin or modality of propagation; they closely preceded (0.2-5 sec) and thus appeared to initiate ictal discharges. Only ictal discharges were blocked by the antagonist of the NMDA receptor 3,3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (CPP, 10 microM), whereas the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) abolished all epileptiform activities. The GABA-mediated potentials continued to occur synchronously in all regions even after concomitant application of CNQX and CPP. [K+]o elevations were recorded in the entorhinal cortex during the ictal discharge (peak values = 13.9 +/- 0.9 mM) and the synchronous GABA-mediated potentials (peak values = 4.2 +/- 0.1 mM); the latter increases were presumably attributable to postsynaptic GABAa-receptor activation because they were abolished by DAGO or BMI. Their role in initiating ictal activity was demonstrated by using DAGO, which abolished both GABA-mediated synchronous potentials and ictal discharges. These data indicate that NMDA-mediated ictal discharges induced by 4AP originate in the entorhinal cortex; such a conclusion is in line with clinical evidence obtained in temporal lobe epilepsy patients. 4AP also induces GABA-mediated potentials that spread within the limbic system when excitatory transmission is blocked and may play a role in initiating ictal discharge by increasing [K+]o.